Based on the statistics provided by the American Cancer Society, approximately four million people have died from cancer since 1990, and cancer, after heart disease, is the second leading cause of death in the United States. Treatments of cancer usually include chemotherapy, radiation, hormones, immunotherapy, and surgery. Chemotherapy remains a preferred treatment, especially in cancer types that are in inoperable or metastatic forms.
Many cytotoxic agents, including antimetabolites, antibiotics, alkylating agents, and mitotic inhibitors, are now available in chemotherapy. These agents usually destroy both normal and tumor cells. It is desirable to develop an antitumor agent that preferentially destroys tumor cells over normal cells.
Due to their pathological conditions, tumor cells differ from normal cells in that their surrounding blood vessels are poorly organized, resulting in inefficient delivery of oxygen to the tumor site. In other words, tumor cells are hypoxic (oxygen deficient). This unique physiology opens the door to the design of cytotoxic agents that are specific for tumor cells.
An aspect of this invention relates to a cytotoxic compound which consists of three components: (1) a proactive alkylating moiety containing an electron-withdrawing group; (2) a bioreductive moiety containing at least two double bonds; and (3) a linker joining together the proactive alkylating moiety and the bioreductive moiety. A xe2x80x9cproactive alkylating moietyxe2x80x9d refers to a functional group which, once activated, replaces an active hydrogen atom of another compound, such as DNA, with one of its alkyl groups in a covalent manner. A xe2x80x9cbioreductive moietyxe2x80x9d refers to a moiety that is capable of undergoing an in vivo reduction (electron-accepting reaction), i.e., bioreduction. The double bonds of the bioreductive moiety, either by themselves, or together with that of the linker, form a conjugated system. The conjugated system allows electrons to flow from the bioreductive moiety to the electron-withdrawing group of the proactive alkylating moiety upon reduction of the bioreductive moiety. This results in breaking the bond between the electron-withdrawing group and the linker and converting the proactive alkylating moiety into an active alkylating compound.
An example of the proactive alkylating moiety is an aromatic group (e.g., phenyl group or naphthyl) substituted with an electron-withdrawing group (e.g., ester, urethane, or carbonate) and a bis(haloethyl)amino group (e.g., a bis(chloroethyl)amino group or nitrogen mustard). The bis(haloethyl)amino group, upon bioreduction, becomes an alkylating group. When the aromatic moiety is a phenyl, each of the two substituents is preferred to be at a meta or para position with respect to each other. Each of the remaining positions of the phenyl, independently, is optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, amino, aminoalkyl, hydroxyl, hydroxylalkyl, alkoxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkoxy, oligoalkylene glycol, amido, ester, aralkoxycarbonylamino, ureido, thio, alkylthio, arylthio, or heteroarylthio. Among them, alkyl, alkoxy, oligoalkylene glycol, aryloxy, heteroaryloxy, and amino are preferred. It is preferable that each of these substituents is at an ortho position with respect to the bis(haloethyl)amino group.
The bioreductive moiety is converted into a second alkylating agent upon bioreduction. Some examples of the bioreductive moiety are 1,4-benzoquinone (i.e., quinone), nitrobenzene, or 1,2-dioxocyclohex-3,5-diene. When the bioreductive moiety is quinone, each of the non-oxo positions of the quinone ring, independently, is optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, amino, aminoalkyl, hydroxyl, hydroxylalkyl, alkoxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkoxy, carboxylate, acyloxyalkyl, ester, amido, amidoalkyl, sulfoamido, sulfonylamino, thio, alkylthio, arylthio, aralkylthio, heteroarylthio, or heteroaralkylthio. The preferred substituents are alkyl, amino, aminoalkyl, alkoxy, hydroxylalkyl, and acyloxyalkyl. If both 2-C and 3-C positions or both 5-C and 6-C positions of the quinone are substituted, the two substituents optionally together form a ring. Two fused rings can be formed with the quinone ring if all non-oxo positions of the quinone are substituted and each pair of the substituents together form a fused ring. The fused ring can be either aliphatic or aromatic. It is also optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, amino, aminoalkyl, hydroxyl, hydroxylalkyl, alkoxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkoxy, carboxylate, acyloxyalkyl, ester, amido, amidoalkyl, sulfoamido, sulfonylamino, thio, alkylthio, arylthio, aralkylthio, heteroarylthio, or heteroaralkylthio. The fused ring optionally contains 1-3 heteroatoms, such as nitrogen, oxygen, or sulfur.
The linker which links the proactive alkylating moiety and the bioreductive moiety together can be one of the following: a methylene group, a C3 hydrocarbon chain containing a double bond, or a C5 hydrocarbon chain containing two alternate double bonds. This linker is optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or oligoalkylene glycol. If the linker contains more than two substituents, two of them can join together to form a 5-6 membered ring. The ring can be aliphatic or aromatic and is optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or oligoalkylene glycol. One to three heteroatoms such as nitrogen, oxygen, or sulfur, can form part of the ring.
A salt of a cytotoxic compound is also within the scope of this invention. For example, the salt can be formed between an amino substituent of a cytotoxic compound and a negatively charged counterion. Suitable counterions include, but are not limited to, chloride, hydrochloride, bromide, iodide, sulfate, nitrate, phosphate, or acetate. Likewise, a negatively charged substituent, e.g., carboxylate, of a compound of this invention can also form a salt with a cation, e.g., an alkali metal cation such as sodium ion or potassium ion; an alkaline earth metal cation such as magnesium cation or calcium cation; or an ammonium cation that can be substitued with one or more organic groups such as tetramethylammonium ion or diisopropylethylammonium ion.
The term xe2x80x9calkylxe2x80x9d in this disclosure denotes a straight or branched hydrocarbon chain containing 1 to 8 carbon atoms, or cyclic hydrocarbon chain containing 3 to 8 carbon atoms. The cyclic hydrocarbon chain may contain 1-3 heteroatoms such as nitrogen, oxygen, or sulfur and may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isopentyl, hexyl, isohexyl, heptyl, octyl, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, isobornyl, cyclohexylmethyl, 1- or 2-cyclohexylethyl, 1-, 2-, or 3-cyclohexylpropyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolindinyl groups.
By the term xe2x80x9calkenylxe2x80x9d is meant a straight or branched hydrocarbon chain containing 2 to 8 carbon atoms or cyclic hydrocarbon chain, i.e., xe2x80x9ccycloalkenyl,xe2x80x9d containing 3 to 8 carbon atoms, which is characterized by having one or more double bonds. The cycloalkenyl may contain 1-3 heteroatoms such as nitrogen, oxygen, or sulfur, i.e., xe2x80x9cheterocycloalkenyl,xe2x80x9d and may also contain fused rings. Typically alkenyl groups include allyl, 2-butenyl, 2-pentenyl, 2-hexenyl, cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclo-octenyl, and norbornylenyl.
xe2x80x9cArylxe2x80x9d is an cyclic aromatic moiety containing 3-8 carbon atoms and may also contain fused rings. Fused aryl denotes an aromatic ring that shares a common carbon-carbon bond with another cyclic moiety. This cyclic moiety can be either an aryl, a cycloalkyl, or a heterocycloalkyl. Typically aryl groups include phenyl, 1-naphthyl, 2-naphthyl, biphenyl, phenanthryl, and anthracyl groups. xe2x80x9cHeteroarylxe2x80x9d refers to aryl groups that contains 1-3 heteroatoms. Typically heterocyclic aromatic rings including coumarinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl and benzthiazolyl. An example of the aralkyl group is 2-phenylethyl.
The term xe2x80x9coligoalkylene glycolxe2x80x9d refers to a chain of 2-5 alkoxy groups. Each of the alkoxy groups may or may not be identical. An example of an oligoalkylene glycol is ethoxymethoxy.
As used herein, substituents such as amino, amido, ester, sulfoamido, sulfonylamino, and ureido are either unsubstituted or substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Further, a divalent substituent such as amido or ester can be connected to its two neighboring moieties in either orientation. The substituents of a cyclic group, e.g., phenyl, can be attached at any available position.
Another aspect of this invention relates to a composition which contains one of the cytotoxic compounds (or its salt) described above and a pharmaceutically acceptable carrier. The compound is in an amount which is effective for treating tumors. Still another aspect of this invention relates to a method of treating tumors, which comprises administering to a patient in need thereof an effective amount of such a cytotoxic compound or its salt. Some examples of tumors which can be treated by this method are leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer. The use of such a cytotoxic compound for the manufacture of a medicament for treating the above-mentioned tumors is also within the scope of this invention.
Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments, and also from the appending claims.
The present invention relates to a cytotoxic compound which has (1) a proactive alkylating moiety containing an electron-withdrawing group and (2) a bioreductive moiety.
Examples of the bioreductive moiety include: 
Examples of the proactive alkylating moiety include: 
Note that in both of the above examples, the ester group of the proactive alkylating moiety is the electron-withdrawing group.
As mentioned above, a cytotoxic compound disclosed in this invention is capable of converting into two alkylating agents upon bioreduction. The mechanism of this conversion can generally be divided into two stages. A cytotoxic compound containing quinone as the bioreductive moiety, methylene group as the linker, and bis(chloroethyl)-amino-phenyl ester as the proactive alkylating moiety is used as an example in the following description.
The first stage involves the reduction of the bioreductive moiety. Typically, this is achieved by cellular enzymes, e.g., cytochrome P450 reductase. Quinone can undergo bioreduction in two one-electron steps, which produces a semiquinone radical anion in the first seduction, and a hydroquinone in the second one. The semiquinone radical anion is very reactive towards oxygen. Indeed, in normal tissues where there is an abundance of oxygen, most of the radical anions are re-oxidized back to quinone. 
As mentioned in the background section, a tumor site is characterized by its poorly organized vascular system, which results in a generally more hypoxic (oxygen-deficient) environment in comparison to that of normal tissues. In other words, reduced compounds are less likely to encounter molecular oxygen and be re-oxidized. Semiquinone radical anions, therefore, have longer halflife and can be further reduced to produce hydroquinone.
In the second stage, a pair of electrons travel from the oxygen of the hydroquinone to the quinone ring (see illustration shown below, where the electron-withdrawing group is xe2x80x94Oxe2x80x94(Cxe2x95x90O)xe2x80x94 (ester) and the linker is a xe2x80x94CH2xe2x80x94 (methylene)), and finally to the oxygen of the electron-withdrawing group of the proactive alkylating moiety via the linker which joins the bioreductive and the proactive alkylating moieties. This electron travelling activity thus results in cleavage of the bond between the electron-withdrawing group and the linker, thereby converting the quinone moiety into a quinone methide. Quinone methides are known alkylating agents capable of attacking nucleophiles, e.g., DNA (See Lin et al., J. Med. Chem. 1972, 15, 127; J. Med. Chem. 1973, 16, 1268; J. Med. Chem. 197, 17, 688; J. Med. Chem. 1975, 18, 917; J. Med. Chem. 1976, 19, 1336). quinone methide 
As a consequence of this bond cleavage, the electron-withdrawing group is converted into one that is much less electron-withdrawing. This conversion, in turn, increases the electron density of the bis(haloethyl)amino group and converts the proactive alkylating moiety into an alkylating agent. Using an ester group as an example, its strong electron-withdrawing character, as indicated by the Hammet substitution constants ("sgr"p=0.45 and "sgr"m=0.37) keeps the bis(chloroethyl)amino alkylating moiety in a deactivated stage. As the ester group is converted into a carboxylate, which is much less electron-withdrawing (with "sgr"p=0 and "sgr"m=xe2x88x920.1), the electron density of the amino nitrogen of the bis(haloethyl)amino increases, thus resulting in a boost in its alkylating activities.
A class of cytotoxic compounds of this invention is represented by formula (I) below: 
wherein each of A, B, C, and D, independently, is xe2x80x94R1, xe2x80x94Rxe2x80x94NR1R2, xe2x80x94Oxe2x80x94R1, xe2x80x94Rxe2x80x94OH, xe2x80x94C(xe2x95x90O)Oxe2x80x94R1, xe2x80x94Rxe2x80x94Oxe2x80x94C(xe2x95x90O)R1, xe2x80x94C(xe2x95x90O)xe2x80x94NR1R2, xe2x80x94Rxe2x80x94NR1xe2x80x94C (xe2x95x90O)R2, xe2x80x94SO2xe2x80x94NR1R2, xe2x80x94Nxe2x95x90SO2, xe2x80x94Sxe2x80x94R1, or xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X) 2. Optionally, A and B together form a 5-6 membered fused ring with the quinone ring, if none of A and B is xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X) 2. Similarly, C and D optionally join together to form a 5-6 membered fused ring with the quinone ring, if none of C and D is xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X)2. The fused ring optionally contains 1-3 heteroatoms such as nitrogen, oxygen, or sulfur, and can optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, xe2x80x94Rxe2x80x94NR1R2, xe2x80x94Oxe2x80x94R1, xe2x80x94Rxe2x80x94OH, xe2x80x94C(xe2x95x90O)Oxe2x80x94R1, xe2x80x94Rxe2x80x94Oxe2x80x94C(xe2x95x90O)R1, xe2x80x94C(xe2x95x90O) xe2x80x94NR1R2, xe2x80x94Rxe2x80x94NR1xe2x80x94C(xe2x95x90O)R2 , xe2x80x94SO2xe2x80x94NR1R2, xe2x80x94Nxe2x95x90SO2 , or xe2x80x94Sxe2x80x94R1. Each R, independently, is alkyl or deleted. Each of R1 and R2, independently, is hydrogen, alkyl, alkenyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. L is xe2x80x94(CR3xe2x95x90CR4)nxe2x80x94CR5R6xe2x80x94, in which each of R3, R4, R5, and R6, independently, is hydrogen, alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or xe2x80x94(O-alkyl)1-5; and n is 0, 1, or 2. The term xe2x80x9cxe2x80x94(O-alkyl)1-5xe2x80x9d refers to an alkoxy group (xe2x80x9cxe2x80x94(O-alkyl)1xe2x80x9d) or an oligoalkylene glycol group (xe2x80x9cxe2x80x94(O-alkyl)2-5xe2x80x9d). R3 and R4, when n is not 0, optionally form a 5- to 6-membered ring together. The ring can be aliphatic or aromatic and can optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or xe2x80x94(O-alkyl)1-5. 1-3 heteroatoms, e.g., nitrogen, oxygen, or sulfur, can also form part of the ring. W is xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94NR1xe2x80x94, or xe2x80x94Oxe2x80x94(xe2x95x90O)Oxe2x80x94. Ph is a phenyl group, optionally substituted with alkyl, alkenyl, aryl, aralkyl, heteroaryl, heteroaralkyl, xe2x80x94Rxe2x80x94NR1R2, xe2x80x94OH, xe2x80x94(O-alkyl)1-5, xe2x80x94O-aryl, xe2x80x94O-aralkyl, xe2x80x94O-heteroaryl, xe2x80x94O-heteroaralkyl, xe2x80x94Rxe2x80x94OH, xe2x80x94C(xe2x95x90O)Oxe2x80x94R1, xe2x80x94Oxe2x80x94C(xe2x95x90O)R1, xe2x80x94C(xe2x95x90O)xe2x80x94NR1R2, xe2x80x94NR1xe2x80x94C(xe2x95x90O)R2, xe2x80x94NR1xe2x80x94C(xe2x95x90O)Oxe2x80x94R2, xe2x80x94NR1xe2x80x94C(xe2x95x90O)NR1R2, or xe2x80x94Sxe2x80x94R1. X is a halo, e.g., fluoro, chloro, bromo, or iodo.
Note that if neither A and B, nor C and D, form a fused ring with the quinone ring, then at least one of A, B, C, or D is xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X)2. Further, if none of A, B, C, and D is xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X)2, then A and B, or C and D (including A and B, as well as C and D) together form a fused ring with the quinone ring. The fused ring (or at least one of the two fused rings if two fused rings are present) contains a double bond between two ring atoms and is substituted with xe2x80x94Lxe2x80x94Wxe2x80x94Phxe2x80x94N(CH2CH2X)2 at one of the two ring atoms. This double bond, together with the double bonds of the quinone ring, form a conjugated system to allow electron to flow from one double bond to another.
Some specific examples of a compound of formula (I) are shown below. 
The preparation of a compound of formula (I) is generally divided into three parts: (1) the preparation of a bioreductive quinone moiety; (2) the preparation of a bis(haloethyl)amino-phenyl moiety; and (3) coupling of the bioreductive quinone moiety and the bix(haloethyl)amino-phenyl moiety. The general synthetic procedures of parts (1)-(3) are described below:
(1) Preparation of a quinone-ring containing bioreductive moiety:
A leaving group, e.g., a halide, that is attached to the linker of a properly protected bioreductive moiety is necessary to couple to a desired bis(haloethyl)amino- containing phenyl moiety in part (3). The leaving group and the linker can be introduced at a non-oxo position of the quinone ring by, e.g., electrophilic substitution reaction. As illustrated in part (1) of Example 1, a hydroxymethyl group resulted at the C2 carbon of 3,5,6-trimethyl-hydroquinone dimethyl ester as the hydroquinone reacted with paraformaldehyde. Since the reaction took place in hydrochloric acid, the hydroxylmethyl reacted further and resulted in the hydroxyl group being replaced with chloride ion. This reaction thus produced a chloromethyl-substituted quinone. The two methyl ester protecting groups were then be deprotected afterwards by hydrolysis.
(2) Preparation of a bis(haloethyl)amino-containing phenyl moiety (chloro is the halo in the following description):
A bis(chloroethyl)amino phenyl moiety can be prepared from, e.g., a nitrobenzoic acid. The carboxylate can be protected in the form of an ester. Suitable substituents to the benzene ring can be coupled to or transformed at this point, e.g., see part (2) of Example 1. The nitro group can then be reduced to form an amino group. This amino group can then react with an ethylene oxide, forming a disubstituted hydroxyethyl amino group. The alkylating moiety, i.e., the bis(chloroethyl)amino moiety, is finally formed when a chlorination agent, e.g., thionyl chloride, is added to the bis(hydroxyethyl)amino-containing intermediate. Similar to the deprotection reaction in part (1), the ester group is being cleaved by hydrolysis.
(3) Coupling reaction of the quinone ring-containing bioreductive moiety and the bis(chloroethyl)amino-containing phenyl moiety:
In a typical example, a quinone ring-containing moiety, e.g., 2-chloromethyl-3,5,6-trimethylbenzoquinone in Example 1, can be coupled to a bis(chloroethyl)amino-containing phenyl moiety, e.g., 3-[bis-(2-chloroethyl)amino-4-methoxybenzoic acid, via a nucleophilic substitution reaction. The carboxylate, which acts as a nucleophile, displaces the halide ion and results in the formation of an ester linkage.
As mentioned above, a pharmaceutical composition of this invention containing a cytotoxic compound in an effective amount can be used to treat tumors. Also within the scope of this invention is a method of treating tumor by administering to a patient such a composition. An effective amount of a cytotoxic compound (or a salt of the cytotoxic compound) is defined as the amount of the compound which, upon administration to a patient in need, confers a therapeutic effect on treated patient. The effective amount to be administered to a patient is typically based on age, surface area, weight, and conditions of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 1966, 50, 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a cytotoxic compound used to practice the invention can range from about 0.1 mg/kg to about 250 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments including use of other antitumor agents and radiation therapy.
The pharmaceutical composition may be administered via the parenteral route, including orally, topically, subcutaneously, intraperitoneally, intramuscularly, and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient. Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
A cytotoxic compound of this invention can also be formulated into dosage forms for other routes of administration utilizing well-known methods. The pharmaceutical composition can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal or a tablet. Capsules may comprise any well-known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with the conventional procedure by compressing mixtures of the active compounds of the present invention and a solid carrier, and a lubricant. Examples of solid carriers include starch and sugar bentonite. The cytotoxic compound can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder and a conventional filler and a tableting agent.
The antitumor activity of the compounds of this invention can be preliminarily evaluated by using a tumor growth regression assay which assesses the ability of tested compounds to inhibit the growth of established solid tumors in mice. The assay can be performed by implanting tumor cells into the fat pads of nude mice. Tumor cells are then allowed to grow to a certain size before the cytotoxic compounds are administered. The volumes of tumor are then monitored for a set number of weeks, e.g., three weeks. General health of the tested animals are also monitored during the course of the assay.
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific examples, which described syntheses and biological testings of various compounds of the present invention, are therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever, All publications recited herein, including patents, are hereby incorporated by reference in their entirety.
Each of the examples 1-7 depicts in detail the synthesis of seven cytotoxic compounds of this invention. Each example is divided into three parts: (1) the preparation of a bioreductive quinone moiety, (2) the preparation of a bis(chloroethyl)amino-phenyl moiety, and (3) the coupling reaction of these two moieties.